Anticancer agent

ABSTRACT

According to the present invention, an anticancer agent is provided that has as an active ingredient thereof a human antibody light chain that demonstrates cytotoxicity against cancer cells and particularly lung cancer cells. The anticancer agent of the present invention primarily comprises: a human antibody κ-type light chain in the form of a dimer in which the variable region is composed of a polypeptide represented by the amino acid sequence of SEQ ID NO: 1, 9 or 13 or an amino acid sequence in which one or a plurality of amino acids have been added, deleted or substituted in these amino acid sequences; or, a human antibody κ-type light chain in the form of a monomer in which the variable region is composed of a polypeptide represented by the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence in which one or a plurality of amino acids have been added, deleted or substituted in the amino acid sequence.

TECHNICAL FIELD

The present invention relates to an anticancer agent containing a humanantibody κ-type light chain that demonstrates cytotoxicity againstcancer cells and particularly lung cancer cells.

The present application claims priority on the basis of Japanese PatentApplication No. 2012-52334, filed in Japan on Mar. 8, 2012, the contentsof which are incorporated herein by reference.

BACKGROUND ART

Antibodies are composed of heavy chains (H chains) and light chains (Lchains). The heavy chains and light chains are composed of a variableregion (VR) and a constant region (CR), and the variable region has acomplementarity determining region (CDR). Moreover, antibody lightchains are classified into κ chains and λ chains.

In recent years, attention has been focused on antibodies having anenzyme-like activity, namely, antibody enzymes. Since antibody enzymeshave both the ability of antibodies to accurately recognize moleculesand the activity of enzymes, they are expected to be applied in numerousfields, including medicine, the chemical industry and the food industry.In particular, since antibody enzymes exhibit high specificity for atarget molecule and are able to impair target molecules due to theirenzyme activity, they are expected to serve as superior anticanceragents that demonstrate few adverse side effects.

The inventors of the present invention have heretofore conducted variousinnovative research on antibody enzymes (see, for example, PatentDocument 1). Antibody enzymes having complete human sequences haveconventionally been unable to be obtained with the exception of theBence-Jones Protein (BJP) obtained from multiple myeloma patients. Sincethere are few multiple myeloma patients and only a small amount of BJPthat has enzyme activity, it was difficult to acquire a human antibodyenzyme. However, since human antibody enzymes are predicted todemonstrate few adverse side effects when administered to a human body,pharmaceutical companies both at home and overseas are awaiting thedevelopment of a useful human antibody enzyme.

PRIOR ART DOCUMENTS Patent Documents

Patent Document 1: Japanese Unexamined Patent Application, FirstPublication No. 2006-197930

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

An object of the present invention is to provide an anticancer agentthat has for an active ingredient thereof a human antibody light chainthat demonstrates cytotoxicity against cancer cells and particularlyagainst lung cancer cells.

Means for Solving the Problems

The inventors of the present invention acquired a novel human antibodylight chain from peripheral blood obtained from volunteershyperimmunized over a plurality of times using a rabies vaccine virus,and as a result of studying those volunteers, surprisingly found thatseveral of the resulting human antibody κ-type light chains demonstrateda high degree of cytotoxicity against cancer cells and particularly lungcancer cells, thereby leading to completion of the present invention.

Namely, the anticancer agent according to the present invention ischaracterized in that it contains:

(1) a human antibody κ-type light chain in the form of a dimer in whichthe variable region is composed of a polypeptide represented by theamino acid sequence of SEQ ID NO: 1, an amino acid sequence in which oneor a plurality of the amino acids in that amino acid sequence have beensubstituted, added or deleted, or an amino acid sequence having homologyof 95% or more with that amino acid sequence;

(2) a human antibody κ-type light chain in the form of a dimer in whichthe variable region is composed of a polypeptide represented by theamino acid sequence of SEQ ID NO: 7, an amino acid sequence in which oneor a plurality of the amino acids in that amino acid sequence have beensubstituted, added or deleted, or an amino acid sequence having homologyof 95% or more with that amino acid sequence;

(3) a human antibody κ-type light chain in the form of a dimer in whichthe variable region is composed of a polypeptide represented by theamino acid sequence of SEQ ID NO: 9, an amino acid sequence in which oneor a plurality of the amino acids in that amino acid sequence have beensubstituted, added or deleted, or an amino acid sequence having homologyof 95% or more with that amino acid sequence;

(4) a human antibody κ-type light chain in the form of a dimer in whichthe variable region is composed of a polypeptide represented by theamino acid sequence of SEQ ID NO: 13, an amino acid sequence in whichone or a plurality of the amino acids in that amino acid sequence havebeen substituted, added or deleted, or an amino acid sequence havinghomology of 95% or more with that amino acid sequence;

(5) a human antibody κ-type light chain in the form of a monomer inwhich the variable region is composed of a polypeptide represented bythe amino acid sequence of SEQ ID NO: 19, an amino acid sequence inwhich one or a plurality of the amino acids in that amino acid sequencehave been substituted, added or deleted, or an amino acid sequencehaving homology of 95% or more with that amino acid sequence;

(6) a human antibody κ-type light chain in the form of a monomer inwhich the variable region is composed of a polypeptide represented by anamino acid sequence consisting of the 1st to 113th amino acids of SEQ IDNO: 38, an amino acid sequence in which one or a plurality of the aminoacids in that amino acid sequence have been substituted, added ordeleted, or an amino acid sequence having homology of 95% or more withthat amino acid sequence;

(7) a human antibody κ-type light chain in the form of a dimer in whichthe variable region is composed of a polypeptide represented by an aminoacid sequence consisting of the 1st to 112th amino acids of SEQ ID NO:40, an amino acid sequence in which one or a plurality of the aminoacids in that amino acid sequence have been substituted, added ordeleted, or an amino acid sequence having homology of 95% or more withthat amino acid sequence; or

(8) a human antibody κ-type light chain in the form of a dimer in whichthe variable region is composed of a polypeptide represented by an aminoacid sequence consisting of the 1st to 107th amino acids of SEQ ID NO:41, an amino acid sequence in which one or a plurality of the aminoacids in that amino acid sequence have been substituted, added ordeleted, or an amino acid sequence having homology of 95% or more withthat amino acid sequence.

Effects of the Invention

According to the present invention, an anticancer agent can be providedthat is highly cytotoxic against cancer cells and particularly lungcancer cells. Since the anticancer agent of the present invention has anantibody enzyme for the active ingredient thereof, it is highly specificfor cancer cells. Moreover, since the amino acid sequence of theantibody enzyme is completely human, it is free of problems such asallergies with respect to humans. Consequently, the anticancer agent ofthe present invention is extremely useful as a highly active, innovativeand novel pharmaceutical and as a test piece for the developmentthereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram indicating amino acid sequences of wild type humanantibody κ-type light chains.

FIG. 2( a) schematically indicates a cDNA design for obtaining a monomerhuman antibody light chain, while FIG. 2 (b) schematically indicates thecompositions of a human antibody light chain prior to introduction of amutation and a human antibody light chain following introduction of amutation.

FIG. 3 is a diagram indicating the results of purifying a polypeptide ofa clone #1, with FIG. 3( a) indicating the results of newly carrying outprimary purification of the polypeptide of clone #1, and FIG. 3( b)indicating the results of newly carrying out secondary purification ofthe polypeptide of clone #1.

FIG. 4 is a graph indicating the results of investigating thecytotoxicity of various clones against cancer cells.

FIG. 5 is a graph indicating the results of investigating thecytotoxicity of various clones against cancer cells.

FIG. 6A is a diagram indicating the amino acid sequences of wild typehuman antibody κ-type light chains.

FIG. 6B is a diagram indicating the amino acid sequences of wild typehuman antibody κ-type light chains.

FIG. 6C is a diagram indicating the amino acid sequences of wild typehuman antibody κ-type light chains continuing from FIG. 6B.

FIG. 6D is a diagram indicating the amino acid sequences of wild typehuman antibody κ-type light chains.

FIG. 6E is a diagram indicating the amino acid sequences of wild typehuman antibody κ-type light chains continuing from FIG. 6D.

FIG. 6F is a diagram indicating the amino acid sequences of wild typehuman antibody κ-type light chains continuing from FIG. 6E.

FIG. 6G is a diagram indicating the amino acid sequences of wild typehuman antibody κ-type light chains.

FIG. 6H is a diagram indicating the amino acid sequences of wild typehuman antibody κ-type light chains continuing from FIG. 6G.

FIG. 6I is a diagram indicating the amino acid sequence of a wild typehuman antibody κ-type light chain.

FIG. 7 is a diagram indicating the results of an in vivo assay.

FIG. 8 is a diagram indicating the results of safety studies (toxicitystudies).

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention provides an anticancer agent containing a humanantibody κ-type light chain that demonstrates cytotoxicity againstcancer cells. In the description of the present application, a “humanantibody κ-type light chain” refers to a κ-type light chain ofhuman-derived immunoglobulin.

In the description of the present application, an “anticancer agent”refers to a pharmaceutical agent having an activity that eradicatescancer cells or suppresses or inhibits the proliferation thereof.

In addition, in the description of the present application,“cytotoxicity” refers to a property that induces cell death or causesfunctional impairment in cells.

More specifically, the active ingredient of the anticancer agentaccording to the present invention in the form of a human antibodyκ-type light chain (to also be referred to as the “human antibody κ-typelight chain according to the present invention”) is any of thosedescribed in (1) to (8) below.

(1) A human antibody κ-type light chain in the form of a dimer in whichthe variable region is composed of a polypeptide represented by theamino acid sequence of SEQ ID NO: 1, an amino acid sequence in which oneor a plurality of the amino acids in that amino acid sequence have beensubstituted, added or deleted, or an amino acid sequence having homologyof 95% or more with that amino acid sequence.

(2) A human antibody κ-type light chain in the form of a dimer in whichthe variable region is composed of a polypeptide represented by theamino acid sequence of SEQ ID NO: 7, an amino acid sequence in which oneor a plurality of the amino acids in that amino acid sequence have beensubstituted, added or deleted, or an amino acid sequence having homologyof 95% or more with that amino acid sequence.

(3) A human antibody κ-type light chain in the form of a dimer in whichthe variable region is composed of a polypeptide represented by theamino acid sequence of SEQ ID NO: 9, an amino acid sequence in which oneor a plurality of the amino acids in that amino acid sequence have beensubstituted, added or deleted, or an amino acid sequence having homologyof 95% or more with that amino acid sequence.

(4) A human antibody κ-type light chain in the form of a dimer in whichthe variable region is composed of a polypeptide represented by theamino acid sequence of SEQ ID NO: 13, an amino acid sequence in whichone or a plurality of the amino acids in that amino acid sequence havebeen substituted, added or deleted, or an amino acid sequence havinghomology of 95% or more with that amino acid sequence.

(5) A human antibody κ-type light chain in the form of a monomer inwhich the variable region is composed of a polypeptide represented bythe amino acid sequence of SEQ ID NO: 19, an amino acid sequence inwhich one or a plurality of the amino acids in that amino acid sequencehave been substituted, added or deleted, or an amino acid sequencehaving homology of 95% or more with that amino acid sequence.

(6) A human antibody κ-type light chain in the form of a monomer inwhich the variable region is composed of a polypeptide represented by anamino acid sequence consisting of the 1st to 113th amino acids of SEQ IDNO: 38, an amino acid sequence in which one or a plurality of the aminoacids in that amino acid sequence have been substituted, added ordeleted, or an amino acid sequence having homology of 95% or more withthat amino acid sequence.

(7) A human antibody κ-type light chain in the form of a dimer in whichthe variable region is composed of a polypeptide represented by an aminoacid sequence consisting of the 1st to 112th amino acids of SEQ ID NO:40, an amino acid sequence in which one or a plurality of the aminoacids in that amino acid sequence have been substituted, added ordeleted, or an amino acid sequence having homology of 95% or more withthat amino acid sequence.

(8) A human antibody κ-type light chain in the form of a dimer in whichthe variable region is composed of a polypeptide represented by an aminoacid sequence consisting of the 1st to 107th amino acids of SEQ ID NO:41, an amino acid sequence in which one or a plurality of the aminoacids in that amino acid sequence have been substituted, added ordeleted, or an amino acid sequence having homology of 95% or more withthat amino acid sequence.

The human antibody κ-type light chain in the form of a dimer in whichthe variable region is composed of a polypeptide represented by theamino acid sequence of SEQ ID NO: 1 may also be referred to as humanantibody κ-type light chain (#1). The human antibody κ-type light chain(#1) can have a known human antibody constant region added to theaforementioned variable region, and in one embodiment, the entire lengthof the amino acid sequence is as shown in SEQ ID NO: 2. CDR1 in thehuman antibody κ-type light chain (#1) consists of the 24th to 39thamino acids in the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO:2, CDR2 consists of the 55th to 61st amino acids in the amino acidsequences of SEQ ID NO: 1 and SEQ ID NO: 2, and CDR3 consists of the94th to 102nd amino acids in the amino acid sequences of SEQ ID NO: 1and SEQ ID NO: 2.

A cysteine residue for forming a disulfide bond is present in a wildtype antibody κ-type light chain that results in the formation of adimer. The human antibody κ-type light chain (#1) also has a cysteineresidue for forming a disulfide bond with another light chain in thesame manner as the wild type. For example, in the case the humanantibody κ-type light chain (#1) is composed of a polypeptiderepresented by the amino acid sequence of SEQ ID NO: 2, then thecysteine residue is the cysteine residue at position 220 in the aminoacid sequence of SEQ ID NO: 2.

As will be subsequently indicated in the examples, the human antibodyκ-type light chain (#1) demonstrates cytotoxicity against cancer cellsand particularly lung cancer cells. Consequently, it is preferable foruse as an active ingredient of an anticancer agent. Since the ability toaccurately recognize a target molecule is important for the humanantibody κ-type light chain (#1) to demonstrate anticancer activity, theactive center of the anticancer activity of the human antibody κ-typelight chain (#1) is in the variable region.

Being able to easily modify several amino acids among amino acidresidues composing a polypeptide without having a significant effect onthe structure or function of the polypeptide is commonly known in theart. Moreover, in addition to artificial modification, mutants are alsocommonly known to exist in naturally-occurring proteins that do notcause a significant change in the structure or function of that protein.Furthermore, in the description of the present application, thesubstitution, addition or deletion of one or a plurality of amino acidsin a specific amino acid sequence X is referred to as mutation.

The human antibody κ-type light chain according to the present inventionmay form a dimer in which the variable region is composed of apolypeptide represented by an amino acid sequence in which one or aplurality of amino acids in the amino acid sequence of SEQ ID NO: 1 havebeen substituted, added or deleted, or an amino acid sequence havinghomology of 95% or more with that amino acid sequence. This polypeptidemay also be referred to as a mutant of the human antibody κ-type lightchain (#1). A mutant of the human antibody κ-type light chain (#1) mayalso be composed of a polypeptide represented by an amino acid sequencein which one or a plurality of amino acids other than the cysteine atposition 220 in the amino acid sequence of SEQ ID NO: 2 have beensubstituted, added or deleted, or an amino acid sequence having homologyof 95% or more with that amino acid sequence.

A mutant of the human antibody κ-type light chain (#1) used as the humanantibody κ-type light chain according to the present invention is adimer having an anticancer action in the same manner as the humanantibody κ-type light chain (#1). Consequently, CDR1, CDR2 and CDR3 of amutant of the human antibody κ-type light chain (#1) are identical tothe amino acid sequence of SEQ ID NO:1 or SEQ ID NO: 2 (are preservedtherein), and the cysteine corresponding to cysteine at position 220 inthe amino acid sequence of SEQ ID NO: 2 is also preserved. In otherwords, a mutant of the human antibody κ-type light chain (#1) ispreferably such that amino acids in regions other than CDR1, CDR2 andCDR3 are mutated and amino acids in other regions of the variable regionare mutated.

A human antibody κ-type light chain in the form of a dimer in which thevariable region is composed of a polypeptide represented by the aminoacid sequence of SEQ ID NO: 9 may also be referred to as human antibodyκ-type light chain (#4). The human antibody κ-type light chain (#4) canhave a known human antibody constant region added to the aforementionedvariable region, and in one embodiment, the entire length of the aminoacid sequence is as shown in SEQ ID NO: 10. CDR1 in the human antibodyκ-type light chain (#4) consists of the 24th to 40th amino acids in theamino acid sequences of SEQ ID NO: 9 and SEQ ID NO: 10, CDR2 consists ofthe 56th to 62nd amino acids in the amino acid sequences of SEQ ID NO: 9and SEQ ID NO: 10, and CDR3 consists of the 95th to 102nd amino acids inthe amino acid sequences of SEQ ID NO: 9 and SEQ ID NO: 10. In addition,a cysteine residue for forming a disulfide bond with another light chainis the cysteine residue at position 220 in the amino acid sequence ofSEQ ID NO: 10.

As will be subsequently indicated in the examples, the human antibodyκ-type light chain (#4) demonstrates cytotoxicity against cancer cellsand particularly lung cancer cells. Consequently, it is preferable foruse as an active ingredient of an anticancer agent.

The human antibody κ-type light chain according to the present inventionmay forma dimer in which the variable region is composed of apolypeptide represented by an amino acid sequence in which one or aplurality of amino acids in the amino acid sequence of SEQ ID NO: 9 havebeen substituted, added or deleted, or an amino acid sequence havinghomology of 95% or more with that amino acid sequence. This polypeptidemay also be referred to as a mutant of the human antibody κ-type lightchain (#4). A mutant of the human antibody κ-type light chain (#4) mayalso be composed of a polypeptide represented by an amino acid sequencein which one or a plurality of amino acids other than the cysteine atposition 220 in the amino acid sequence of SEQ ID NO: 10 have beensubstituted, added or deleted, or an amino acid sequence having homologyof 95% or more with that amino acid sequence.

A mutant of the human antibody κ-type light chain (#4) used as the humanantibody κ-type light chain according to the present invention is adimer having an anticancer action in the same manner as the humanantibody κ-type light chain (#4). Consequently, CDR1, CDR2 and CDR3 of amutant of the human antibody κ-type light chain (#4) are identical tothe amino acid sequence of SEQ ID NO:9 or SEQ ID NO: 10 (are preservedtherein), and the cysteine corresponding to cysteine at position 220 inthe amino acid sequence of SEQ ID NO: 10 is also preserved. In otherwords, a mutant of the human antibody κ-type light chain (#4) ispreferably such that amino acids in regions other than CDR1, CDR2 andCDR3 are mutated and amino acids in other regions of the variable regionare mutated.

A human antibody κ-type light chain in the form of a dimer in which thevariable region is composed of a polypeptide represented by the aminoacid sequence of SEQ ID NO: 13 may also be referred to as human antibodyκ-type light chain (#7). The human antibody κ-type light chain (#7) canhave a known human antibody constant region added to the aforementionedvariable region, and in one embodiment, the entire length of the aminoacid sequence is as shown in SEQ ID NO: 14. CDR1 in the human antibodyκ-type light chain (#7) consists of the 24th to 39th amino acids in theamino acid sequences of SEQ ID NO: 13 and SEQ ID NO: 14, CDR2 consistsof the 55th to 61st amino acids in the amino acid sequences of SEQ IDNO: 13 and SEQ ID NO: 14, and CDR3 consists of the 94th to 101st aminoacids in the amino acid sequences of SEQ ID NO: 13 and SEQ ID NO: 14. Inaddition, a cysteine residue for forming a disulfide bond with anotherlight chain is the cysteine residue at position 219 in the amino acidsequence of SEQ ID NO: 14.

As will be subsequently indicated in the examples, the human antibodyκ-type light chain (#7) demonstrates cytotoxicity against cancer cellsand particularly lung cancer cells. Consequently, it is preferable foruse as an active ingredient of an anticancer agent.

The human antibody κ-type light chain according to the present inventionmay form a dimer in which the variable region is composed of apolypeptide represented by an amino acid sequence in which one or aplurality of amino acids in the amino acid sequence of SEQ ID NO: 13have been substituted, added or deleted, or an amino acid sequencehaving homology of 95% or more with that amino acid sequence. Thispolypeptide may also be referred to as a mutant of the human antibodyκ-type light chain (#7). A mutant of the human antibody κ-type lightchain (#7) may also be composed of a polypeptide represented by an aminoacid sequence in which one or a plurality of amino acids other than thecysteine at position 219 in the amino acid sequence of SEQ ID NO: 14have been substituted, added or deleted, or an amino acid sequencehaving homology of 95% or more with that amino acid sequence.

A mutant of the human antibody κ-type light chain (#7) used as the humanantibody κ-type light chain according to the present invention is adimer having an anticancer action in the same manner as the humanantibody κ-type light chain (#7). Consequently, CDR1, CDR2 and CDR3 of amutant of the human antibody κ-type light chain (#7) are identical tothe amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14 (are preservedtherein), and the cysteine corresponding to cysteine at position 219 inthe amino acid sequence of SEQ ID NO: 14 is also preserved. In otherwords, a mutant of the human antibody κ-type light chain (#7) ispreferably such that amino acids in regions other than CDR1, CDR2 andCDR3 are mutated and amino acids in other regions of the variable regionare mutated.

A human antibody κ-type light chain in the form of a monomer in whichthe variable region is composed of a polypeptide represented by theamino acid sequence of SEQ ID NO: 19 may also be referred to as humanantibody κ-type light chain (22F6_monomer). The human antibody κ-typelight chain (22F6_monomer) can have a known human antibody constantregion added to the aforementioned variable region, and in oneembodiment, the entire length of the amino acid sequence is representedby an amino acid sequence in which the 219th cysteine in the amino acidsequence of SEQ ID NO: 20 has been deleted or substituted with anotheramino acid (such as alanine). CDR1 in the human antibody κ-type lightchain (22F6_monomer) consists of the 24th to 39th amino acids in theamino acid sequences of SEQ ID NO: 19 and SEQ ID NO: 20, CDR2 consistsof the 55th to 61st amino acids in the amino acid sequences of SEQ IDNO: 19 and SEQ ID NO: 20, and CDR3 consists of the 94th to 101st aminoacids in the amino acid sequences of SEQ ID NO: 19 and SEQ ID NO: 20.

As will be subsequently indicated in the examples, the human antibodyκ-type light chain (22F6_monomer) demonstrates cytotoxicity againstcancer cells and particularly lung cancer cells. Consequently, it ispreferable for use as an active ingredient of an anticancer agent.

The human antibody κ-type light chain according to the present inventionmay be a monomer in which the variable region is composed of apolypeptide represented by an amino acid sequence in which one or aplurality of amino acids in the amino acid sequence of SEQ ID NO: 20have been substituted, added or deleted, or an amino acid sequencehaving homology of 95% or more with that amino acid sequence. Thispolypeptide may also be referred to as a mutant of the human antibodyκ-type light chain (22F6_monomer). A mutant of the human antibody κ-typelight chain (22F6_monomer) may also be composed of a polypeptiderepresented by an amino acid sequence in which the 219th cysteine hasbeen deleted or substituted with another amino acid and one or aplurality of amino acids other than the amino acid at position 219 havebeen substituted, added or deleted in the amino acid sequence of SEQ IDNO: 20, or an amino acid sequence having homology of 95% or more withthat amino acid sequence.

A mutant of the human antibody κ-type light chain (22F6_monomer) used asthe human antibody κ-type light chain according to the present inventionis a monomer having an anticancer action in the same manner as the humanantibody κ-type light chain (22F6_monomer). Consequently, CDR1, CDR2 andCDR3 of a mutant of the human antibody κ-type light chain (22F6_monomer)are identical to the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO:20 (are preserved therein), and the cysteine corresponding to cysteineat position 219 in the amino acid sequence of SEQ ID NO: 20 is deletedor substituted with another amino acid. In other words, a mutant of thehuman antibody κ-type light chain (22F6_monomer) is preferably such thatamino acids in regions other than CDR1, CDR2 and CDR3 are mutated andamino acids in other regions of the variable region are mutated.

In addition, the human antibody κ-type light chain according to thepresent invention may also contain an additional polypeptide. Typicalexamples of additional polypeptides include epitope-tagged polypeptidessuch as those tagged with His tag, Myc or Flag.

A person with ordinary skill in the art is able to easily mutate one ora plurality of amino acids among amino acid residues that compose apolypeptide or add an epitope-tagged polypeptide using a knowntechnology. For example, an arbitrary base of a polynucleotide thatencodes a polypeptide can be mutated in accordance with a known pointmutagenesis method. In addition, a primer corresponding to an arbitrarysite of a polynucleotide that encodes a polypeptide can be designed tocreate a deletion mutant or an addition mutant.

The human antibody κ-type light chain according to the present inventionincludes a naturally-occurring purification product, a product obtainedby a chemical synthesis procedure, and a product produced byrecombination technology from a prokaryotic host or eukaryotic host(including bacterial cells, yeast cells, higher plant cells, insectcells and mammalian cells). The human antibody κ-type light chain may ormay not be glycosylated depending on the host used in the recombinantproduction procedure. Moreover, the human antibody κ-type light chainaccording to the present invention can contain a modified initiatingmethionine group in several cases as a result of a host interventionprocess.

Although the human antibody κ-type light chain according to the presentinvention may be a polypeptide in which amino acids are linked bypeptide bonds, it is not limited thereto, and the polypeptide may alsobe a composite polypeptide containing a structure other than that of apolypeptide. As used in the present description, although examples of a“structure other than that of a polypeptide” include sugar chains andisoprenoid groups, there are no particular limitations thereon.

The human antibody κ-type light chain according to the present inventioncan be produced using an expression system known in the art, such as arecombination expression system or a cell-free expression system, byusing a vector containing a polynucleotide encoding the human antibodyκ-type light chain (polypeptide).

In the case of using a recombination expression system, a method can beemployed having the steps of, for example, incorporating apolynucleotide encoding the human antibody κ-type light chain accordingto the present invention into a recombination expression vector followedby introducing into a host enabling expression thereof according to aknown method, translating within the host (transformant) and purifyingthe resulting polypeptide. The recombination expression vector may ormay not be a plasmid, and is only required to enable the targetpolynucleotide to be introduced into the host.

In the case of introducing an exogenous polynucleotide into a host inthis manner, a promoter that functions in the host so as to expressexogenous polynucleotides is preferably incorporated into the expressionvector. Although the method used to purify the recombinantly producedpolypeptide varies according to the properties of the host andpolypeptide used, a target polypeptide can be purified comparativelyeasily using a tag and the like.

In the case of using a cell-free expression system (cell-free proteinsynthesis system), a polynucleotide encoding the human antibody κ-typelight chain according to the present invention is preferably added to asolution containing components such as ribosomes or t-RNA required forprotein translation and synthesis followed by incubating at a suitabletemperature and purifying the synthesized polypeptide.

Examples of cell-free protein synthesis systems include systems usingwheat germ extract, systems using rabbit reticulocyte extract, systemsusing E. coli S30 extract and systems using cell component extractsobtained from plant devacuolated protoplasts. In general, althougheukaryotic cell systems, namely, systems using wheat germ extract orsystems using rabbit reticulocyte extract, are selected for translationof eukaryotic genes, the aforementioned synthesis system is selected inconsideration of such factors as the origin of the gene to be translated(prokaryotic or eukaryotic) or the purpose for which the protein is tobe used following synthesis. Various commercially available kits can beused for these synthesis systems.

Furthermore, since various viral gene products frequently expressactivity by going through a complex biochemical reaction involving thecytomembrane, such as the endoplasmic reticulum or Golgi bodies,following translation, it is necessary to add cytomembrane components(such as microsomal membrane) in order to reproduce the variousbiochemical reactions in vitro. Cell component extracts obtained fromplant devacuolated protoplasts are preferable since they can be used asa cell-free protein synthesis liquid that retains cytomembranecomponents, thereby eliminating the need to add microsomal membrane.

As used in the present description, “cytomembrane components” areintended to refer to cell organelles composed of lipid membrane presentin the cytomembrane (namely, all types of intracellular granules such asendoplasmic reticulum, Golgi bodies, mitochondria, chloroplast andvacuoles). In particular, since endoplasmic reticulum and Golgi bodiesfulfill an important role in post-translation modification of proteins,they are essential cell components for maturation of membrane proteinsand secretory proteins.

Human antibody κ-type light chain synthesized with a host expressionsystem or a cell-free protein synthesis system is preferably purified.Although a step for purifying human antibody κ-type light chain ispreferably a step in which a cell extract is prepared from cells ortissue using a known method (such as a method in which the cells ortissue is homogenized, followed by centrifuging and recovering thesoluble fraction), followed by purifying the human antibody κ-type lightchain from this cell extract using a known method (such as ammoniumsulfate precipitation or ethanol precipitation, acid extraction, anionicor cationic chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxyapatitechromatography or lectin chromatography), it is not limited thereto.High-performance liquid chromatography (HPLC) is most preferably usedfor purification.

In addition, the human antibody κ-type light chain according to thepresent invention can also be purified from cells or tissues thatexpress the human antibody κ-type light chain in nature. For example,cells or tissues that express the human antibody κ-type light chainaccording to the present invention in nature can be identified using anantibody or an oligonucleotide. Purification of a human antibody κ-typelight chain from cells or tissue can also be carried out in the samemanner as in the case of purifying a human antibody κ-type light chainsynthesized using a host expression system and the like.

In addition, the human antibody κ-type light chain according to thepresent invention can also be chemically synthesized. There are noparticular limitations on the chemical synthesis method, and may becarried out by any method used when chemically synthesizingpolypeptides.

The anticancer agent according to the present invention has the humanantibody κ-type light chain according to the present invention as anactive ingredient thereof. Although the mechanism of action by which thehuman antibody κ-type light chain according to the present inventiondemonstrates cytotoxicity against cancer cells has not been completelydetermined, it is presumed that, as a result of the human antibodyκ-type light chain according to the present invention specificallyrecognizing and binding to a specific molecule or structure on thesurface of cancer cells simultaneous to decomposing a portion of thecomponents of cancer cells by utilizing its own enzyme activity, thefunction of the cancer cells is impaired, proliferation thereof isinhibited or cell death is induced.

The anticancer agent according to the present invention can be injectedor administered directly for use in humans or animals. The anticanceragent according to the present invention can also be formulated forparenteral administration, mucosal administration, intramuscularadministration, intravenous administration, subcutaneous administration,intraocular administration or transcutaneous administration.

Typically, protein contained in a composition can be administered at adose of 0.01 mg/kg to 30 mg/kg of body weight, preferably at 0.1 mg/kgto 10 mg/kg of body weight, and even more preferably at 0.1 mg/kg to 1mg/kg of body weight.

The anticancer agent according to the present invention can also containa pharmaceutically acceptable carrier, diluent or vehicle (includingcombinations thereof) in addition to the human antibody κ-type lightchain according to the present invention. Pharmaceutically acceptablecarriers or vehicles for therapeutic use are commonly known in the fieldof pharmacy, and are described in, for example, Remington'sPharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro, ed., 1985).Pharmaceutically usable carriers, vehicles or diluents can be suitablyselected by a person with ordinary skill in the art in accordance withthe intended administration route and standard pharmaceutical practices.In addition, the anticancer agent according to the present invention canfurther contain an arbitrary suitable binder, lubricant, suspensionagent, coating agent or solubilizing agent.

Conditions required for composition and/or formulation can varydepending on the use of different delivery systems. As an examplethereof, the anticancer agent according to the present invention can beformulated so as to be delivered using a minipump, by a mucosal route inthe form of, for example, a nasal spray or aerosol for inhalation, orfor parenteral delivery (here, the anticancer agent according to thepresent invention is formulated in an injectable form for delivery via,for example, an intravenous route, an intramuscular route or asubcutaneous route). Alternatively, the formula can be designed so as tobe delivered by both routes. For example, the anticancer agent accordingto the present invention demonstrates a high level of cytotoxicityagainst lung cancer cells in particular. Consequently, the anticanceragent according to the present invention is preferably in the form of anasal spray or aerosol for inhalation that enables it to be efficientlydelivered to pneumocytes from the nose or bronchi.

In addition, in the case of using the anticancer agent according to thepresent invention in an application in which it is administered into thebody, various technologies can be used for improving the stability(half-life in blood) of the active ingredient in the form of the humanantibody κ-type light chain in the body. For example, the half-life inthe blood of antibodies such as IgG is known to be prolonged if neonatalFc receptor (FcRn) is bound to the Fc region (see, for example,Roopenian, D. C., et al., Nat. Rev. Immunol., Vol. 7, 715-725 (2007)),and the C-terminal of the human antibody κ-type light chain according tothe present invention can be modified so as to have binding activitywith FcRn. In addition, the human antibody κ-type light chain accordingto the present invention can be in the form of a dimer, and polyethyleneglycol (PEG) can be added as well.

The anticancer agent according to the present invention can beincorporated in a kit, for example, together with instructions and thelike on the form in which it is to be administered. The kit can alsocontain various other pharmaceuticals that can be used with theanticancer agent according to the present invention.

In addition, since the anticancer agent according to the presentinvention has for the active ingredient thereof an antibody κ-type lightchain that is highly effective in recognizing a target molecule, it doesnot demonstrate cytotoxicity against cancer cells in which the targetmolecule of the antibody light chain is not present on the cell surfacethereof. Consequently, the anticancer agent of the present invention isexpected to be useful in distinguishing types of cancer.

EXAMPLES

Although the following provides a more detailed explanation of thepresent invention through examples thereof, the present invention is notlimited by these examples.

Example 1 1. Preparation of Human Peripheral Blood cDNA

Lymphocytes were isolated using Ficoll-paque from peripheral bloodacquired from volunteers hyperimmunized over a plurality of times usingrabies virus vaccine. Total RNA was obtained from roughly 3.0×10⁷isolated lymphocytes using an RNA extraction kit (Stratagene Corp.). Thetarget cDNA (cDNA library) was then prepared by reverse transcribing thetotal RNA with the ThermoScript RT-PCT System (Invitrogen Inc.) usingoligo (dT) as primer.

2. Acquisition of Human Antibody κ-Type Light Chain Genes

PCR reactions were carried out in two stages using the cDNA acquired instep 1 above as template and using primers for amplifying antibody lightchain gene having a Vκ gene belonging to subgroup II to obtain roughly750 bp PCR products (κ-type light chain genes belonging to subgroup II).These PCR products were cloned and subjected to sequence analysis, andthe Vκ gene in each germline gene was estimated by a homology search. Asa result, all of the resulting 18 clones belonged to subgroup II. Amongthese, nine clones, namely, clone #1 (germline genotype: A18b), clone #2(germline genotype: A3/A19), clone #4 (germline genotype: 011/o1), clone#7 (germline genotype: A3/A19), clone #8 (germline genotype: A18b),clone #9 (germline genotype: A18b), clone #11 (germline genotype: A18b),clone #13 (germline genotype: A3/A19) and clone #14 (germline genotype:A3/A19) were used in subsequent experimentation.

3. Expression of Human Antibody κ-Type Light Chains

Each of the clones acquired in step 2 above was respectively introducedinto a plasmid vector having an His tag sequence site followed byintroducing the plasmid vector into Escherichia coli to producetransformants. When each transformant was cultured and subjected toinduction of expression with IPTG, the protein expressed in the E. coliwas able to be identified as a human antibody light chain by SDS-PAGEanalysis and Western blotting using anti-human (Fab′)₂ antibody. Theresulting human antibody light chains had M (initiating methionine) onthe N-terminal and LEHHHHHH (SEQ ID NO: 23) derived from the plasmidvector on the C-terminal.

4. Preparation of Human Peripheral Blood cDNA

Subjects were hyperimmunized over a plurality of times using rabiesvirus vaccine followed by measurement of serum neutralizing activity.Peripheral blood was collected from the donor subject having the highestlevel of serum neutralizing activity (7.21 U), and lymphocytes wereisolated from the peripheral blood using Ficoll-paque. Total RNA wasthen obtained from roughly 3.0×10⁷ isolated lymphocytes using an RNAextraction kit (Stratagene Corp.). cDNA to be used as template wasprepared in a PCR reaction to be subsequently described by reversetranscribing the total RNA with the ThermoScript RT-PCR System(Invitrogen Inc.) using oligo(dT) as primer.

5. Acquisition of Human Antibody κ-Type Light Chain Genes

A PCR reaction was carried out using a primer set for comprehensivelyamplifying human antibody light chain gene and using the cDNA acquiredin step 4 above as template to obtain a roughly 660 bp PCR product. ThisPCR product was purified and inserted into the E. coli expression vectorpET101/D-TOPO® (Invitrogen Inc.) to construct an LCA library.Furthermore, protein in which an His tag was added to the C-terminal ofthe protein encoded by the PCR product was expressed from an expressionvector in which the PCR product was inserted in the pET101/D-TOPOvector. PCR reactions were carried out using the cDNA of this LCAlibrary as template and using primers for amplifying human antibodylight chain gene having a Vκ gene belonging to subgroup II to obtainroughly 660 bp PCR products. These PCR products were cloned andsubjected to sequence analysis and their amino acid sequences and lightchain variable and constant regions were estimated using analyticalsoftware (Genetix® Ver. 8) followed by estimation of the Vκ gene in eachgermline gene. Among these clones, two clones, namely, clone 22F6(germline genotype: A3/A19) and clone 23D4 (germline genotype: A3/A19)were used in subsequent experimentation. The resulting human antibodylight chains had M (initiating methionine) on the N-terminal andLEHHHHHH (SEQ ID NO: 23) derived from the plasmid vector on theC-terminal.

As a result of sequencing each clone, the total length of the humanantibody light chain pertaining to clone #1 (human antibody light chain(#1_WT)) was the base sequence indicated in SEQ ID NO: 27, the totallength of the human antibody light chain pertaining to clone #8 (humanantibody light chain (#8_WT)) was the base sequence indicated in SEQ IDNO: 28, the total length of the human antibody light chain pertaining toclone #9 (human antibody light chain (#9_WT)) was the base sequenceindicated in SEQ ID NO: 29, the total length of the human antibody lightchain pertaining to clone #11 (human antibody light chain (#11_WT)) wasthe base sequence indicated in SEQ ID NO: 30, the total length of thehuman antibody light chain pertaining to clone #4 (human antibody lightchain (#4_WT)) was the base sequence indicated in SEQ ID NO: 31, thetotal length of the human antibody light chain pertaining to clone #2(human antibody light chain (#2_WT)) was the base sequence indicated inSEQ ID NO: 32, the total length of the human antibody light chainpertaining to clone #7 (human antibody light chain (#7_WT)) was the basesequence indicated in SEQ ID NO: 33, the total length of the humanantibody light chain pertaining to clone #13 (human antibody light chain(#13_WT)) was the base sequence indicated in SEQ ID NO: 34, the totallength of the human antibody light chain pertaining to clone #14 (humanantibody light chain (#14_WT)) was the base sequence indicated in SEQ IDNO: 35, the total length of the human antibody light chain pertaining toclone 22F6 (human antibody light chain (22F6_WT)) was the base sequenceindicated in SEQ ID NO: 36, and the total length of the human antibodylight chain pertaining to clone 23D4 (human antibody light chain(23D4_WT)) was the base sequence indicated in SEQ ID NO: 37.

The amino acid sequences estimated from each of the base sequences areshown in FIG. 1. In addition, the locations of the variable regions,constant regions and CDR1 to CDR3 are also shown. The human antibodylight chain pertaining to clone #1 (human antibody light chain (#1_WT))was the amino acid sequence shown in SEQ ID NO: 2, the human antibodylight chain pertaining to clone #8 (human antibody light chain (#8_WT))was the amino acid sequence shown in SEQ ID NO: 4, the human antibodylight chain pertaining to clone #9 (human antibody light chain (#9_WT))was the amino acid sequence shown in SEQ ID NO: 6, the human antibodylight chain pertaining to clone #11 (human antibody light chain(#11_WT)) was the amino acid sequence shown in SEQ ID NO: 8, the humanantibody light chain pertaining to clone #4 (human antibody light chain(#4_WT)) was the amino acid sequence shown in SEQ ID NO: 10, the humanantibody light chain pertaining to clone #2 (human antibody light chain(#2_WT)) was the amino acid sequence shown in SEQ ID NO: 12, the humanantibody light chain pertaining to clone #7 (human antibody light chain(#7_WT)) was the amino acid sequence shown in SEQ ID NO: 14, the humanantibody light chain pertaining to clone #13 (human antibody light chain(#13_WT)) was the amino acid sequence shown in SEQ ID NO: 16, the humanantibody light chain pertaining to clone #14 (human antibody light chain(#14_WT)) was the amino acid sequence shown in SEQ ID NO: 18, the humanantibody light chain pertaining to clone 22F6 (human antibody lightchain (22F6_WT)) was the amino acid sequence shown in SEQ ID NO: 20, andthe human antibody light chain pertaining to clone 23D4 (human antibodylight chain (23D4_WT)) was the amino acid sequence shown in SEQ ID NO:22.

Furthermore, the wild type human antibody light chains used in thepresent example were polypeptides in which methionine was added to theN-terminal of each amino acid sequence shown in FIG. 1 and LEHHHHHH (SEQID NO: 23) derived from the plasmid vector was added to the C-terminal.

6. Production of Monomer Human Antibody Light Chains

The human antibody κ-type light chains of the clones acquired in steps 2and 5 above formed dimers due to the formation of disulfide (S—S) bondsby cysteine on the C-terminal. Then, cDNA was designed so as to formonly monomer human antibody enzymes by introducing a mutation in whichthe cysteine involved in S—S bond formation (cysteine on the C-terminalof the amino acid sequences of FIG. 1) is substituted with alanine. Thedetails of this design with respect to the human antibody light chainhaving LEHHHHHH derived from the plasmid vector on the C-terminalthereof (#1_WT) are shown in FIG. 2. As shown in FIG. 2( a), TGTencoding cysteine at position 220 in the full-length human antibodyenzyme gene is substituted with GCT. As a result, as shown in FIG. 2(b), although a dimer is formed in the original amino acid sequence dueto the presence of cysteine at position 220, S—S bonds are not formed inthe substituted amino acid sequence as a result of substituting alanineat position 220, thereby resulting in a monomer.

More specifically, TGT encoding the aforementioned cysteine in thewild-type full-length human antibody enzyme gene was substituted withGCTCTCGAGCACCACCACCACCACCACTGA (SEQ ID NO: 26) that encodes ALEHHHHHH(SEQ ID NO: 25) (having a stop codon). In other words, the monomer humanantibody light chain used in the present example was a polypeptide inwhich methionine was added to the N-terminal of each amino acid sequenceshown in FIG. 1 and ALEHHHHHH was added to the C-terminal instead ofcysteine. Furthermore, among those mutants obtained in this manner, inwhich the cysteine involved in S—S bonding was substituted with alanine,the mutant of human antibody light chain (#1_WT) is referred to as thehuman antibody light chain (#1_C220A), the mutant of human antibodylight chain (#8_WT) is referred to as the human antibody light chain(#8_C220A), the mutant of human antibody light chain (#9_WT) is referredto as the human antibody light chain (#9_C220A), the mutant of humanantibody light chain (#11_WT) is referred to as the human antibody lightchain (#11_C220A), the mutant of human antibody light chain (#4_WT) isreferred to as the human antibody light chain (#4_C220A), the mutant ofhuman antibody light chain (#2_WT) is referred to as the human antibodylight chain (#2_C220A), the mutant of human antibody light chain (#7_WT)is referred to as the human antibody light chain (#7_C220A), the mutantof human antibody light chain (#13_WT) is referred to as the humanantibody light chain (#13_C220A), the mutant of human antibody lightchain (#14_WT) is referred to as the human antibody light chain(#14_C220A), the mutant of human antibody light chain (22F6_WT) isreferred to as the human antibody light chain (22F6_C220A) and themutant of human antibody light chain (23D4_WT) is referred to as thehuman antibody light chain (23D4_C220A).

7. Purification of Human Antibody Light Chains

Each of the human antibody light chains was subjected to primarypurification and secondary purification in the manner described below.FIG. 3( a) is a diagram indicating the results of Ni-NTA columnchromatography and SDS-PAGE analysis during primary purification ofhuman antibody light chain (#1_WT) and human antibody light chain(#1_C220A). FIG. 3( b) is a diagram indicating the results of cationexchange chromatography and SDS-PAGE analysis during secondarypurification.

As shown on the left side of FIG. 3( a), buffer A (25 mM Tris-HCl (pH8.0), 0.25 M NaCl, 40 mM imidazole and 0.005% Tween 20) was passedthrough the column after applying the sample until all the flow-throughfraction had passed through the column. As indicated by the broken linein the graph on the left side, the concentration of imidazole wasincreased gradually from 40 mM to 300 mM to elute a component bound tothe gel. An Ni-NTA agarose column (diameter: 1 cm, 2 ml) was used forthe column and the flow rate was maintained at 0.1 mL/min throughoutpurification. As shown on the right side of FIG. 3( a), a target band ofroughly 31 kDa was detected in fractions 30 to 37. These samples werecombined and subjected to the secondary purification indicated below.

As shown on the left side of FIG. 3( b), buffer A (50 mM sodium acetate(pH 5.4), 0.2 M NaCl and 0.005% Tween 20) was passed through the columnafter applying the sample until all the flow-through fraction had passedthrough the column. As indicated by the broken line in the graph on theleft side, the concentration of NaCl was increased gradually from 0.2 Mto 0.4 M to elute a component bound to the gel. The SP5PW column (ToshoCorp.) was used for the column and the flow rate was maintained at 0.1ml/min throughout purification. Components contained in the sample priorto purification, the region “a” surrounded by broken lines in the graph(fraction numbers 10 to 15) and the region “c” surrounded by brokenlines in the graph (fraction numbers 25 to 30) were analyzed bySDS-PAGE. As shown on the right side of FIG. 3( b), a target band ofroughly 31 kDa was detected in regions “a” and “c” in the reducedsample. In addition, in the unreduced sample, a roughly 31 kDa band wasdetected only in region “a” while a roughly 51 kDa band was detectedonly in region “c”. As has been described above, the monomer of theantibody light chain is roughly 31 kDa and the dimer is roughly 51 kDa.Sample a is the monomer fraction of the antibody light chain whilesample c is the dimer fraction of the antibody light chain.

The other clones also contained dimers and monomers in the expressionproducts of the wild-type human antibody light chains in the same manneras clone (#1), dimers were purified by two-stage purification utilizingNi-NTA column chromatography and cation exchange chromatography,monomers were contained in the expression products of mutants in whichcysteine involved in S—S bonding had been mutated to alanine, and themonomers were purified by the same two-stage purification.

8. Cytotoxicity Against Cancer Cells

A test was conducted of the cytotoxicity of various human antibodyκ-type light chains against cancer cells. Human alveolar adenocarcinomacell line A549 purchased from ATCC was used for the cancer cells, andthe cells were cultured in accordance with routine methods using F-12Kmedium containing 10% fetal calf serum (FCS).

First, after thawing and recovering frozen A549 cells, 100 μl aliquotsof the cells were disseminated in a 96-well plate to a concentration of5×10³ cells/well. After culturing for 24 hours at 37° C. and removingthe medium added to the 96-well plate by decantation, each humanantibody κ-type light chain adjusted to a concentration of about 1 mg/mLwas added in 100 μL aliquots to each well. 10 μl aliquots of WST-1reagent (Roche Diagnostics GmbH) were added to each well at 24 hours and48 hours after adding the human antibody κ-type light chains (48 hoursand 72 hours after disseminating the cells), followed by measurement ofabsorbance of the formasan pigment formed (Abs 450 nm) 1, 1.5 and 2hours later. Cell viability was determined in each well based on theresulting absorbance results using a value of 100% for cell viability ina well to which a human antibody κ-type light chain was not added (N.C.)followed by evaluation of cytotoxicity of the added human antibodyκ-type light chains.

Cell viability at 24 hours and 48 hours after adding human antibodyκ-type light chain is shown in FIG. 4, FIG. 5, Table 1 and Table 2 foreach of the human antibody κ-type light chains. The results for theclones having a germline genotype of A18b or 011/o1 are shown in FIG. 4and Table 1, and the results for the clones having a germline genotypeof A3/A19 are shown in FIG. 5 and Table 2. In addition, Tables 1 and 2also indicate the concentrations of the human antibody κ-type lightchains in the wells.

TABLE 1 Cell viability (%) Concentration After After Clone in well (μM)24 hr 48 hr #1_WT 44 60 60 #1 C220A 40 72 75 #8 WT 29 79 81 #8 C220A 3896 93 #9 WT 20 78 75 #9 C220A 40 86 84 #11 WT 28 73 80 #11 C220A 44 9594 #4 WT 20 50 54 #4 C220A 44 96 93

TABLE 2 Cell viability (%) Concentration After After Clone in well (μM)24 hr 48 hr #2 WT 27 81 86 #2 C220A 40 94 95 #7 WT 35 49 53 #7 C220A 4092 93 #13 WT 40 101 101 #13 C220A 56 97 81 #14 WT 32 100 94 #14 C220A 4089 88 22F6 WT 62 80 92 22F6 C220A 32 65 64 23D4 WT 28 83 85 23D4 C220A44 96 92

As a result, the four clones consisting of clone (#1_WT), clone (#4_WT),clone (#7_WT) and clone (22F6_C220A) demonstrated cytotoxicity on theorder of 40% to 50% against A549 cells. Other clones were observed tohardly demonstrate any cytotoxicity against A549 cells.

Among these four clones, clone (#4_WT) and clone (#7_WT) demonstratedparticularly potent cytotoxicity. Among these, clone (#7_WT), namely,human antibody κ-type light chain (#7), was suggested to have an effectthat suppresses proliferation of A549 cells since there were hardly anychanges in the number of cells in the wells between prior to addition ofthe human antibody κ-type light chain (0 hours) and after addition ofthe human antibody κ-type light chain (48 hours).

In addition, on the basis of the results for the clones used in thistest, potent cytotoxicity was suggested to be present in dimers since itwas observed that dimers (WT) have a tendency to demonstrate more potentcytotoxicity than monomers.

In addition, cytotoxicity of human antibody κ-type light chain againstvarious cell lines was evaluated in the same manner as described abovewhile also including other clones. Those results are shown in Table 3.

TABLE 3 Cell viability (%) 24 hr after 48 hr after Cell type Cloneaddition addition A549 #1 H31Y C220A 72 53 #7 VL(I) 77 82 #7 RLI 74 90C51 78 87 C87 75 65 MOLT-4 #1 H31Y C220A 57.1 60.2 #4 wt 73.8 90.6 #7 EI87 80.2 #7 TR 93.2 74.1 #7 RLI 55 73 #7 VL 77.3 84.2 S13 75.3 108 S2178.2 91.3 S38 77.3 85.5 C51 59.4 63 ES-2 #1 H31Y C220A 59.9 72.7 #4 83.993.3 #7 wt 98.3 98.6 #7 RLI 100 94.8 #10 79.9 92.4 #11 57 78.8 22F6 63.189 22F6 C220A 53.2 67.4 C51 71.2 70.7 C67 69.7 76.1 C82 62.2 72.7 C8878.1 76.6 BxPC #4 58 63.3 #7 G 88.3 71.9 #7 EI 80.5 69.9 #7 RLI 87.977.3 #7 VL 77.7 75.9 #13 120.4 67.6 #14 116.6 69.3 22F6 115.2 65.5 B-16#7 wt 85 92

As a result, clone (#1_H31Y C220A) demonstrated a high level ofcytotoxicity against A549 cells, MOLT-4 cells and ES-2 cells. Inaddition, clone (#7 RLI) and clone (C51) demonstrated a high level ofcytotoxicity against MOLT-4 cells. Moreover, clone (#4) demonstrated ahigh level of cytotoxicity against ES-2 cells.

Furthermore, the amino acid sequence of clone (#1_H31Y C220A) is shownin SEQ ID NO: 38, the amino acid sequence of clone (#7 VL(I)) is shownin SEQ ID NO: 39, the amino acid sequence of clone (#7 RLI) is shown inSEQ ID NO: 40, the amino acid sequence of clone (C51) is shown in SEQ IDNO: 41, the amino acid sequence of clone (C87) is shown in SEQ ID NO:42, the amino acid sequence of clone (#7 EI) is shown in SEQ ID NO: 43,the amino acid sequence of clone (#7 TR) is shown in SEQ ID NO: 44, theamino acid sequence of clone (#7 VL) is shown in SEQ ID NO: 45, theamino acid sequence of clone (S13) is shown in SEQ ID NO: 46, the aminoacid sequence of clone (S21) is shown in SEQ ID NO: 47, the amino acidsequence of clone (S38) is shown in SEQ ID NO: 48, the amino acidsequence of clone (#10) is shown in SEQ ID NO: 49, the amino acidsequence of clone (C67) is shown in SEQ ID NO: 50, the amino acidsequence of clone (C82) is shown in SEQ ID NO: 51, the amino acidsequence of clone (C88) is shown in SEQ ID NO: 52, and the amino acidsequence of clone (#7 G) is shown in SEQ ID NO: 53.

In addition, as shown in FIGS. 7 and 8, the anticancer agent containingthe human antibody κ-type light chain of the present application did notdemonstrate any toxicity in animal studies.

INDUSTRIAL APPLICABILITY

The present invention allows the development of a novel anticancer agentand the use thereof in the field of cancer treatment.

1. An anticancer composition, comprising: (1) a human antibody κ-typelight chain in the form of a dimer in which a variable region iscomposed of a polypeptide represented by an amino acid sequence of SEQID NO: 1, an amino acid sequence in which one or a plurality of theamino acids in the amino acid sequence have been substituted, added ordeleted, or an amino acid sequence having homology of 95% or more withthe amino acid sequence; (2) a human antibody κ-type light chain in theform of a dimer in which a variable region is composed of a polypeptiderepresented by an amino acid sequence of SEQ ID NO: 7, an amino acidsequence in which one or a plurality of the amino acids in the aminoacid sequence have been substituted, added or deleted, or an amino acidsequence having homology of 95% or more with the amino acid sequence;(3) a human antibody κ-type light chain in the form of a dimer in whicha variable region is composed of a polypeptide represented by an aminoacid sequence of SEQ ID NO: 9, an amino acid sequence in which one or aplurality of the amino acids in the amino acid sequence have beensubstituted, added or deleted, or an amino acid sequence having homologyof 95% or more with the amino acid sequence; (4) a human antibody κ-typelight chain in the form of a dimer in which a variable region iscomposed of a polypeptide represented by an amino acid sequence of SEQID NO: 13, an amino acid sequence in which one or a plurality of theamino acids in the amino acid sequence have been substituted, added ordeleted, or an amino acid sequence having homology of 95% or more withthe amino acid sequence; (5) a human antibody κ-type light chain in theform of a monomer in which a variable region is composed of apolypeptide represented by an amino acid sequence of SEQ ID NO: 19, anamino acid sequence in which one or a plurality of the amino acids inthe amino acid sequence have been substituted, added or deleted, or anamino acid sequence having homology of 95% or more with the amino acidsequence; (6) a human antibody κ-type light chain in the form of amonomer in which a variable region is composed of a polypeptiderepresented by an amino acid sequence consisting of the 1st to 113thamino acids of SEQ ID NO: 38, an amino acid sequence in which one or aplurality of the amino acids in the amino acid sequence have beensubstituted, added or deleted, or an amino acid sequence having homologyof 95% or more with the amino acid sequence; (7) a human antibody κ-typelight chain in the form of a dimer in which a variable region iscomposed of a polypeptide represented by an amino acid sequenceconsisting of the 1st to 112th amino acids of SEQ ID NO: 40, an aminoacid sequence in which one or a plurality of the amino acids in theamino acid sequence have been substituted, added or deleted, or an aminoacid sequence having homology of 95% or more with the amino acidsequence; or (8) a human antibody κ-type light chain in the form of adimer in which a variable region is composed of a polypeptiderepresented by an amino acid sequence consisting of the 1st to 107thamino acids of SEQ ID NO: 41, an amino acid sequence in which one or aplurality of the amino acids in the amino acid sequence have beensubstituted, added or deleted, or an amino acid sequence having homologyof 95% or more with the amino acid sequence; and at least one selectedfrom the group consisting of a pharmaceutically acceptable carrier,diluent and vehicle.
 2. The anticancer composition according to claim 1,wherein the human antibody κ-type light chain of (1) above is a humanantibody κ-type light chain in the form of a dimer composed of apolypeptide represented by an amino acid sequence of SEQ ID NO: 2, anamino acid sequence in which one or a plurality of the amino acids inthe amino acid sequence have been substituted, added or deleted, or anamino acid sequence having homology of 95% or more with the amino acidsequence; the human antibody κ-type light chain of (2) above is a humanantibody κ-type light chain in the form of a dimer composed of apolypeptide represented by an amino acid sequence of SEQ ID NO: 8, anamino acid sequence in which one or a plurality of the amino acids inthe amino acid sequence have been substituted, added or deleted, or anamino acid sequence having homology of 95% or more with the amino acidsequence; the human antibody κ-type light chain of (3) above is a humanantibody κ-type light chain in the form of a dimer composed of apolypeptide represented by an amino acid sequence of SEQ ID NO: 10, anamino acid sequence in which one or a plurality of the amino acids inthe amino acid sequence have been substituted, added or deleted, or anamino acid sequence having homology of 95% or more with the amino acidsequence; the human antibody κ-type light chain of (4) above is a humanantibody κ-type light chain in the form of a dimer composed of apolypeptide represented by an amino acid sequence of SEQ ID NO: 14, anamino acid sequence in which one or a plurality of the amino acids inthe amino acid sequence have been substituted, added or deleted, or anamino acid sequence having homology of 95% or more with the amino acidsequence; the human antibody κ-type light chain of (5) above is a humanantibody κ-type light chain in the form of a monomer composed of apolypeptide represented by an amino acid sequence in which cysteine atposition 219 has been deleted or substituted by an amino acid other thancysteine in the amino acid sequence of SEQ ID NO: 20, an amino acidsequence in which one or a plurality of the amino acids in the aminoacid sequence have been substituted, added or deleted, or an amino acidsequence having homology of 95% or more with the amino acid sequence;the human antibody κ-type light chain of (6) above is a human antibodyκ-type light chain in the form of a monomer composed of a polypeptiderepresented by an amino acid sequence of SEQ ID NO: 38, an amino acidsequence in which one or a plurality of the amino acids in the aminoacid sequence have been substituted, added or deleted, or an amino acidsequence having homology of 95% or more with the amino acid sequence;the human antibody κ-type light chain of (7) above is a human antibodyκ-type light chain in the form of a dimer composed of a polypeptiderepresented by the amino acid sequence of SEQ ID NO: 40, an amino acidsequence in which one or a plurality of the amino acids in the aminoacid sequence have been substituted, added or deleted, or an amino acidsequence having homology of 95% or more with the amino acid sequence; orthe human antibody κ-type light chain of (8) above is a human antibodyκ-type light chain in the form of a dimer composed of a polypeptiderepresented by an amino acid sequence of SEQ ID NO: 41, an amino acidsequence in which one or a plurality of the amino acids in the aminoacid sequence have been substituted, added or deleted, or an amino acidsequence having homology of 95% or more with the amino acid sequence. 3.The anticancer composition according to claim 1, wherein the anticancercomposition is in a form which enables the anticancer composition to bedelivered to pneumocytes from a nose or bronchi.
 4. The anticancercomposition according to claim 1, wherein protein contained in theanticancer composition is a dose of 0.01 mg/kg to 30 mg/kg of bodyweight.
 5. A method of eradicating cancer cells or suppressing orinhibiting a proliferation of the cancer cells, comprising:administering a therapeutically effective amount of an anticancercomposition to an object requiring an administration, the anticancercomposition comprising: (1) a human antibody κ-type light chain in theform of a dimer in which a variable region is composed of a polypeptiderepresented by an amino acid sequence of SEQ ID NO: 1, an amino acidsequence in which one or a plurality of the amino acids in the aminoacid sequence have been substituted, added or deleted, or an amino acidsequence having homology of 95% or more with the amino acid sequence;(2) a human antibody κ-type light chain in the form of a dimer in whicha variable region is composed of a polypeptide represented by an aminoacid sequence of SEQ ID NO: 7, an amino acid sequence in which one or aplurality of the amino acids in the amino acid sequence have beensubstituted, added or deleted, or an amino acid sequence having homologyof 95% or more with the amino acid sequence; (3) a human antibody κ-typelight chain in the form of a dimer in which a variable region iscomposed of a polypeptide represented by an amino acid sequence of SEQID NO: 9, an amino acid sequence in which one or a plurality of theamino acids in the amino acid sequence have been substituted, added ordeleted, or an amino acid sequence having homology of 95% or more withthe amino acid sequence; (4) a human antibody κ-type light chain in theform of a dimer in which a variable region is composed of a polypeptiderepresented by an amino acid sequence of SEQ ID NO: 13, an amino acidsequence in which one or a plurality of the amino acids in the aminoacid sequence have been substituted, added or deleted, or an amino acidsequence having homology of 95% or more with the amino acid sequence;(5) a human antibody κ-type light chain in the form of a monomer inwhich a variable region is composed of a polypeptide represented by anamino acid sequence of SEQ ID NO: 19, an amino acid sequence in whichone or a plurality of the amino acids in the amino acid sequence havebeen substituted, added or deleted, or an amino acid sequence havinghomology of 95% or more with the amino acid sequence; (6) a humanantibody κ-type light chain in the form of a monomer in which a variableregion is composed of a polypeptide represented by an amino acidsequence consisting of the 1st to 113th amino acids of SEQ ID NO: 38, anamino acid sequence in which one or a plurality of the amino acids inthe amino acid sequence have been substituted, added or deleted, or anamino acid sequence having homology of 95% or more with the amino acidsequence; (7) a human antibody κ-type light chain in the form of a dimerin which a variable region is composed of a polypeptide represented byan amino acid sequence consisting of the 1st to 112th amino acids of SEQID NO: 40, an amino acid sequence in which one or a plurality of theamino acids in the amino acid sequence have been substituted, added ordeleted, or an amino acid sequence having homology of 95% or more withthe amino acid sequence; or (8) a human antibody κ-type light chain inthe form of a dimer in which a variable region is composed of apolypeptide represented by an amino acid sequence consisting of the 1stto 107th amino acids of SEQ ID NO: 41, an amino acid sequence in whichone or a plurality of the amino acids in the amino acid sequence havebeen substituted, added or deleted, or an amino acid sequence havinghomology of 95% or more with the amino acid sequence.
 6. A method oferadicating cancer cells or suppressing or inhibiting a proliferation ofthe cancer cells according to claim 5, wherein the human antibody κ-typelight chain of (1) above is a human antibody κ-type light chain in theform of a dimer composed of a polypeptide represented by an amino acidsequence of SEQ ID NO: 2, an amino acid sequence in which one or aplurality of the amino acids in the amino acid sequence have beensubstituted, added or deleted, or an amino acid sequence having homologyof 95% or more with the amino acid sequence; the human antibody κ-typelight chain of (2) above is a human antibody κ-type light chain in theform of a dimer composed of a polypeptide represented by an amino acidsequence of SEQ ID NO: 8, an amino acid sequence in which one or aplurality of the amino acids in the amino acid sequence have beensubstituted, added or deleted, or an amino acid sequence having homologyof 95% or more with the amino acid sequence; the human antibody κ-typelight chain of (3) above is a human antibody κ-type light chain in theform of a dimer composed of a polypeptide represented by an amino acidsequence of SEQ ID NO: 10, an amino acid sequence in which one or aplurality of the amino acids in the amino acid sequence have beensubstituted, added or deleted, or an amino acid sequence having homologyof 95% or more with the amino acid sequence; the human antibody κ-typelight chain of (4) above is a human antibody κ-type light chain in theform of a dimer composed of a polypeptide represented by an amino acidsequence of SEQ ID NO: 14, an amino acid sequence in which one or aplurality of the amino acids in the amino acid sequence have beensubstituted, added or deleted, or an amino acid sequence having homologyof 95% or more with the amino acid sequence; the human antibody κ-typelight chain of (5) above is a human antibody κ-type light chain in theform of a monomer composed of a polypeptide represented by an amino acidsequence in which cysteine at position 219 has been deleted orsubstituted by an amino acid other than cysteine in the amino acidsequence of SEQ ID NO: 20, an amino acid sequence in which one or aplurality of the amino acids in the amino acid sequence have beensubstituted, added or deleted, or an amino acid sequence having homologyof 95% or more with the amino acid sequence; the human antibody κ-typelight chain of (6) above is a human antibody κ-type light chain in theform of a monomer composed of a polypeptide represented by an amino acidsequence of SEQ ID NO: 38, an amino acid sequence in which one or aplurality of the amino acids in the amino acid sequence have beensubstituted, added or deleted, or an amino acid sequence having homologyof 95% or more with the amino acid sequence; the human antibody κ-typelight chain of (7) above is a human antibody κ-type light chain in theform of a dimer composed of a polypeptide represented by the amino acidsequence of SEQ ID NO: 40, an amino acid sequence in which one or aplurality of the amino acids in the amino acid sequence have beensubstituted, added or deleted, or an amino acid sequence having homologyof 95% or more with the amino acid sequence; or the human antibodyκ-type light chain of (8) above is a human antibody κ-type light chainin the form of a dimer composed of a polypeptide represented by an aminoacid sequence of SEQ ID NO: 41, an amino acid sequence in which one or aplurality of the amino acids in the amino acid sequence have beensubstituted, added or deleted, or an amino acid sequence having homologyof 95% or more with the amino acid sequence.
 7. A method of eradicatingcancer cells or suppressing or inhibiting a proliferation of the cancercells according to claim 5, wherein the object requiring theadministration develops lung cancer.
 8. A method of eradicating cancercells or suppressing or inhibiting a proliferation of the cancer cellsaccording to claim 5, wherein the anticancer composition demonstrates acytotoxicity against cancer cells which are present on a cell surface.9. A method of eradicating cancer cells or suppressing or inhibiting aproliferation of the cancer cells according to claim 5, comprising:administrating the anticancer composition in a form which enables theanticancer composition to be delivered to pneumocytes from a nose orbronchi.